Peptide nucleic acids (hereinafter, referred to as ‘PNA’), a kind of artificial DNA analog, were firstly reported wherein the nucleic bases are linked by peptide bonds instead of phosphate bonds (Nielsen P E, Egholm M, Berg R H, Buchardt O, “Sequence-selective recognition of DNA by strand displacement with a thymine-substituted polyamide”, Science 1991, Vol. 254, pp 1497-1500). PNA are not found in nature and synthesized through chemical methods. PNA undergo hybridization reaction with natural nucleic acids having the complementary base sequence to form a double strand. In case of having identical number of the nucleobases, a PNA/DNA double strand is more stable than a DNA/DNA double strand, and a PNA/RNA double strand is more stable than a DNA/RNA double strand. Most commonly used backbone of PNA is N-(2-aminoethyl)glycine repeatedly linked by amide bond, which is electrically neutral differently from that of natural nucleic acids with negative charge.
Four nucleobases in PNA occupy almost the same space as nucleobases of DNA, having almost same distance between nucleobases as that of natural nucleic acids. PNA are more stable chemically and biologically than natural nucleic acids, because they are not decomposed by a nuclease or a protease. The stability of a PNA/DNA or PNA/RNA double strand is not affected by salt concentration because PNA is electrically neutral. Due to these properties, PNA can better recognize the complimentary nucleotide sequence than natural nucleic acids, so that PNA can be applied for diagnosis and other biological or medical purposes.
When a nucleotide sequence is recognized or detected in a homogeneous solution by using a probe of known base sequence, typically one target sequence is recognized at one time. It is difficult to detect more than a few sequences at the same time by using fluorescent dyes of different colors. On the other hand, a lot more number of sequences can be detected concurrently when probes are immobilized on a solid surface. DNA microarrays wherein several hundred thousands of probes are arranged in two dimensions have been already put into practical use. PNA microarrays or PNA chips employing PNA probes instead of DNA probes have been also known (Brandt O, Hoheisel J D, “Peptide nucleic acids on microarrays and other biosensors”, Trends in Biotechnology 2004, Vol. 22, pp 617-622). A method to simultaneously examine a plurality of targets by immobilizing PNA probes on distinguishable microbeads or microspheres of several micrometer size has been reported (Rockenbauer E, Petersen K H, Vogel U, Bolund L, Kølvraa S, Nielsen K V, Nexeø BA, “SNP genotyping using microsphere-linked PNA and flow cytometric detection”, Cytometry Part A 2005, Vol. 64A, pp 80-86). Fluorescence is widely used to determine whether or not hybridization reaction occurs between probes and complementary nucleotide sequences, whereas electrical detection methods also known by using a field-effect transistor with PNA immobilized on silicon semiconductor or silicon nanowire [F. Uslu et al. “Labelfree fully electronic nucleic acid detection system based on a field-effect transistor device”, Biosensors and Bioelectronics 2004, Vol. 19, pp 1723-1731; J. Hahm and C. M. Lieber, “Direct ultrasensitive electrical detection of DNA and DNA sequence variations using nanowire nanosensors”, Nano Letters 2004, Vol. 4, pp 51-54]. A device to recognize nucleotide sequence by detecting impedance change has been also reported [A. Macanovic et al. “Impedance-based detection of DNA sequences using a silicon transducer with PNA as the probe layer”, Nucleic Acids Research 2004, Vol. 32, e20].
Since the mass of the probe is changed after hybridization of the target nucleic acid, the nucleotide sequence can be detected by measuring the mechanical change resulted therefrom. The resonance frequency of a microcantilever or a surface acoustic wave (SAW) sensor changes after hybridization of DNA or RNA, so that it can be use for detection. Microcantilevers and SAW sensors using PNA have been reported [S. Manalis and T. Burg, U.S. Pat. No. 7,282,329 “Suspended microchannel detectors”; P. Warthoe and S. Iben, US Patent Application Publication 2004/0072208 A1 “Surface acoustic wave sensors and method for detecting target analytes”].
In such a device or method of multiplex analysis using PNA probes, the PNA probes needs to be immobilized on solid surface. Covalent bonds formed by a chemical reaction is more stable than physical attraction, and thus frequently selected as a method of immoblilization. Covalent bonds generated by aldehyde-amine, carboxylic acid-amine, or epoxide-amine reaction are widely used in biochips such as PNA microarrays, DNA microarrays and protein micorarrays (M. Schena, Microarray analysis, A. John Wiley & Sons, Inc., 2003, pp 95-120). In order to immobilize PNA on a glass surface, a glass surface is subject to silylation by an organosilane substance having aldehyde, amine or epoxy group in order to expose the functional group on the glass surface. N-terminal amine group of a PNA probe is reacted with the exposed functional group to form a covalent bond.
By using a functional group of high reactivity, the efficiency of immobilization of the probe can be enhanced. At a terminal of DNA or PNA probes, a highly active functional group such as hydrazide may be used (S. Raddatz et al., “Hydrazide oligonucleotides: new chemical modification for chip array attachment and conjugation”, Nucleic Acids Research, 2002, Vol. 30, pp 4793-4802). According to this method, a biochip with higher sensitivity can be obtained by enhancing immobilization efficiency of DNA or PNA probes to a surface. The immobilizing efficiency of probes can be also enhanced by increasing the reactivity on a surface. According to this method, a highly active linker is chemically bonded to a simple amine or aldehyde functional group immobilized on a solid surface to expose activated ester or isothiocyanate, which would then react with DNA or PNA probes. However, such a functional group of high reactivity at a probe terminal or on solid surface is easily degraded and is difficult to keep active.